Interleukin 15 as selectable marker for gene transfer in lymphocytes

ABSTRACT

The present invention relates to the use of interleukin-15 (IL-15) as selectable marker for gene transfer, preferably of at least one gene of therapeutic interest, into a mammalian cell or cell line, in particular a human cell or cell line. The present invention furthermore relates to transgenic mammalian cells or cell lines expressing IL-15 as selectable marker and co-expressing at least one protein of interest encoded by at least one gene of interest, which is preferably a protein of therapeutic interest. The present invention is in particular suitable for chimeric antigen receptors (CARs) as the gene or protein of interest and their expression in lymphocytes. The transgenic mammalian cells and cell lines are furthermore suitable for use as a medicament, in particular in the treatment of cancer and in immunotherapy, such as adoptive, target-cell specific immunotherapy.

This application is a National Stage Application of InternationalApplication Number PCT/EP2011/004491, filed Sep. 6, 2011; which claimspriority to European Patent Application No. 10009346.7, filed Sep. 8,2010; which are incorporated herein by reference in their entirety.

The present invention relates to the use of interleukin-15 (IL-15) asselectable marker for gene transfer, preferably of at least one gene oftherapeutic interest, into a mammalian cell or cell line, in particulara human cell or cell line. The present invention furthermore relates totransgenic mammalian cells or cell lines expressing IL-15 as selectablemarker and co-expressing at least one protein of interest encoded by atleast one gene of interest, which is preferably a protein of therapeuticinterest. The present invention is in particular suitable for chimericantigen receptors (CARs) as the gene or protein of interest and theirexpression in lymphocytes. The transgenic mammalian cells and cell linesare furthermore suitable for use as a medicament, in particular in thetreatment of cancer and in immunotherapy, such as adoptive, target-cellspecific immunotherapy.

BACKGROUND OF THE INVENTION

T lymphocytes recognize specific antigens through interaction of the Tcell receptor (TCR) with short peptides presented by majorhistocompatibility complex (MHC) class I or II molecules. For initialactivation and clonal expansion, naïve T cells are dependent onprofessional antigen-presenting cells (APCs) that provide additionalco-stimulatory signals. TCR activation in the absence of co-stimulationcan result in unresponsiveness and clonal anergy. To bypassimmunization, different approaches for the derivation of cytotoxiceffector cells with grafted recognition specificity have been developed.Chimeric antigen receptors (CARs) have been constructed that consist ofbinding domains derived from natural ligands or antibodies specific forcell-surface antigens, genetically fused to effector molecules such asthe TCR alpha and beta chains, or components of the TCR-associated CD3complex. Upon antigen binding, such chimeric antigen receptors link toendogenous signaling pathways in the effector cell and generateactivating signals similar to those initiated by the TCR complex. Sincethe first reports on chimeric antigen receptors, this concept hassteadily been refined and the molecular design of chimeric receptors hasbeen optimized (for a review see Uherek et al., 2001). Aided by advancesin recombinant antibody technology, chimeric antigen receptors targetedto a wide variety of antigens on the surface of cancer cells and ofcells infected by human immunodeficiency virus (HIV) have been generated(for a review see Uherek et al., 2001).

The expression of CARs with specificity for tumor-associated or viralcell surface antigens in lymphocytes such as T cells or natural killer(NK) cells generates antigen-specific effector cells for the use inadoptive, target-cell specific immunotherapy. Such CARs are composed ofa cell recognition domain such as a scFv antibody fragment forrecognition of a tumor-cell surface antigen fused via a flexible linkerregion to an intracellular signaling domain such as CD3 zeta-chain. CARexpression retargets the cytotoxic activity of lymphocytes to tumorcells that are otherwise resistant to cytolysis by immune effector cells(Uherek et al., 2001; Uherek et al., 2002; Müller et al., 2008; Tavri etal., 2009). Thereby, gene transfer using viral vectors or physicaltransfection methods is of limited efficiency, resulting in only afraction of the cells permanently incorporating and expressing thetransfered gene construct. Hence, it is desirable to include aselectable marker gene in such vector constructs to allow selection andenrichment of gene-modified cells prior to therapeutic applications suchas adoptive therapy.

Depending on the cell type used, the relatively low transductionefficiency of viral vectors employed for genetic modification oflymphocytes (in particular NK cells) with effector genes of therapeuticvalue (such as genes encoding CAR) limits the relative proportion ofgene-modified cells in the transduced cell pool. In principle, inclusionof a selectable marker gene in the vector constructs would allowselection and enrichment of gene-modified cells prior to potentialtherapeutic applications in adoptive immunotherapy. However, availableselection markers such as bacterial resistance genes and bacterialenzymes cannot be used due to their non-human origin and their potentialimmunogenicity. Furthermore, selection using such markers requiresantibiotics or toxic reagents which must be added to the culture medium.

Therefore, the present invention aims to provide means and methods forthe transfer of effector genes of therapeutic interest into mammalian(human) cells, in particular lymphocytes, utilizing a selectable markergene of human origin which allows selective enrichment of gene-modifiedcells in standard culture medium without addition of toxic compounds.

Furthermore, the present invention aims to provide means and methods formedical application(s) of the mammalian (human) cells obtained thereby.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by usinginterleukin-15 (IL-15) or functional equivalents thereof as selectablemarker for/of gene transfer into a mammalian cell or cell line, whereinthe mammalian cell or cell line is selected from effector cells of theimmune system which require cytokines for growth and survival.

Thereby, upon gene transfer (of the IL-15 into said mammalian cell orcell line) the expression of the IL-15 as selectable marker results insurvival or growth of the mammalian cell or cell line in the absence ofsaid cytokines.

According to the present invention this object is furthermore solved bya transgenic mammalian cell or cell line expressing IL-15 as selectablemarker for/of gene transfer and co-expressing at least one protein ofinterest (other than IL-15) encoded by at least one gene of interest(other than IL-15), wherein the mammalian cell or cell line is selectedfrom effector cells of the immune system which require cytokines forgrowth and survival and wherein the expression of the IL-15 asselectable marker results in survival or growth of the mammalian cell orcell line in the absence of said cytokines.

According to the present invention this object is furthermore solved bythe transgenic mammalian cell or cell line of the invention for use as amedicament.

According to the present invention this object is furthermore solved bythe transgenic mammalian cell or cell line of the invention for use inthe treatment of cancer or in immunotherapy.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is tobe understood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art. For the purpose of thepresent invention, all references cited herein are incorporated byreference in their entireties.

Interleukin-15 As Selectable Marker for Gene Transfer

As described above, the present invention provides the use ofinterleukin-15 (IL-15) or functional equivalents thereof as selectablemarker for gene transfer into a mammalian cell or cell line.

Thereby, the expression of said IL-15 as selectable marker results insurvival or growth of the mammalian cell or cell line.

—IL-15

Interleukin 15 (IL-15) belongs to the IL-15/IL-21 family of cytokines.IL-15 has biological activities similar to IL-2, and has been shown tostimulate the growth of natural killer cells, activated peripheral bloodT lymphocytes, tumor infiltrating lymphocytes (TILs), and B cells. Inaddition, IL-15 has also been shown to be a chemoattractant for humanblood T lymphocytes, and to be able to induce lymphokine-activatedkiller (LAK) activity in NK cells and to induce the generation ofcytolytic effector cells. The IL-15 cDNA encodes a 162 amino acid (aa)residue precursor protein containing a 48 aa residue leader that iscleaved to generate the 114 aa residue mature IL-15. In humans,interleukin 15 is encoded by the IL15 gene. Like IL-2, IL-15 binds toand signals through the IL-2/IL-15 beta chain (CD122) and the commongamma chain (gamma-C, CD132). Other cytokines which signal throughreceptor complexes that contain the common gamma chain but employ areceptor beta chain different from that of the IL-15 and IL-2 receptorcomplexes, include IL-4, IL-7, IL-9, and IL-21.

According to the invention, the IL-15 used is human IL-15:

The amino acid sequence of homo sapiens interleukin 15 (IL-15)preproprotein (Genbank Accession No. NP_000576.1):

Amino acid sequence [SEQ ID NO. 1]MRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFSAGLPKTEANW VNVISDLKKI EDLIQSMHID ATLYTESDVHPSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANNSLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS

The protein encoding nucleotide sequence including the translation stopcodon of homo sapiens interleukin 15 (IL-15) cDNA, representingnucleotides 370-858 of interleukin 15 transcript variant 3 (GenbankAccession No. NM_000585.3):

Nucleotide sequence [SEQ ID NO. 2]ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAAC ACTTCTTGA

In an embodiment, the IL-15 nucleic acid sequence can be codon-optimizedfor expression in mammalian cells, preferably for expression in humancells.

According to the invention, the IL-15 used is human IL-15 with the aminoacid sequence of SEQ ID NO. 1 or an amino acid sequence that has atleast 95% sequence identity or 99% sequence identity to the amino acidsequence of SEQ ID NO. 1;

or the IL-15 used is human IL-15 encoded by the nucleotide sequence ofSEQ ID NO. 2 or a nucleotide sequence with at least 95% sequenceidentity or 99% sequence identity to the nucleotide sequence of SEQ IDNO. 2.

Codon-optimization refers to the exchange in a sequence of interest ofcodons that are generally rare in highly expressed genes of a givenspecies by codons that are generally frequent in highly expressed genesof such species, such codons encoding the same amino acids as the codonsthat are being exchanged.

The skilled artisan will be able to design and utilize suitable codonoptimizations of the above sequences.

Within the scope of this invention are also the nucleotide sequencesobtained due to the degeneration of the genetic code of the nucleotidesequences disclosed herein.

The IL-15 is preferably comprised in an expression or gene construct,which is transferred into the mammalian cell or cell line.

An “expression or gene construct” (wherein both terms are usedinterchangeably throughout this specification) refers to a nucleic acidconstruct, usually an expression vector or plasmid, that is used tointroduce a specific gene sequence into a target cell. Once theexpression or gene construct is inside the cell, the protein that isencoded by the gene is produced by the cellular transcription andtranslation machinery. The expression or gene construct is designed tocontain respective regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the construct, including promoter and terminator sequences). The goalof a well-designed expression or gene construct is the production oflarge amounts of stable mRNA, and therefore proteins.

In an embodiment of an expression or gene construct according to thisinvention, a nucleotide sequence encoding (human) IL-15, i.e. a codingsequence of (human) IL-15, and a gene of interest are separated by aregulatory element, preferably an internal ribosome entry site (IRES),enabling their simultaneous expression under the control of a singlepromoter.

The expression or gene construct comprising the IL-15 is preferably a(DNA) plasmid or a viral vector, such as a lentiviral vector, agamma-retroviral vector or an adeno-associated virus vector.

The nucleic acids of this invention comprise DNA (such as dsDNA, ssDNA,cDNA), RNA (such as dsRNA, ssRNA, mRNA), combinations thereof orderivatives (such as PNA) thereof.

The expression or gene construct comprises a nucleotide sequenceencoding (human) IL-15, i.e. a coding sequence of (human) IL-15.

The coding sequence of (human) IL-15 is preferably a cDNA of (human)IL-15.

The coding sequence of IL-15 is preferably

-   -   the nucleotide sequence encoding human IL-15 with the amino acid        sequence of SEQ ID NO. 1 or functional equivalents thereof,    -   the nucleotide sequence comprising or having the nucleotide        sequence of human IL-15 transcript variant 3 of SEQ ID NO. 2 or        functional equivalents thereof,

-   or complementary sequences thereof

-   or codon-optimized sequences thereof,

-   or nucleotide sequences encoding amino acid sequences with at least    95% sequence identity or 99% sequence identity to the amino acid    sequence of SEQ ID NO. 1,

-   or nucleotide sequences with at least 95% sequence identity or 99%    sequence identity to the nucleotide sequence of SEQ ID NO. 2.

The term “functional equivalent” defines a protein or nucleotidesequence, having a different amino acid or base sequence, compared tothe sequences disclosed herein, but exhibiting the same function invitro and in vivo. An example of a functional equivalent is a modifiedor synthetic gene, encoding the expression of a protein identical orhighly homologous to that encoded by the wildtype gene.

A “functional equivalent” of (human) IL-15 refers to a protein that hasan amino acid sequence or nucleotide sequence encoding therefore withless than 100% sequence identity to SEQ ID NO. 1 or 2, respectively, butfunctions as IL-15 inside a cell (the host cell), which means that saidprotein binds to the IL-15 receptor complex and initiates signallingthrough beta and gamma chains of such receptor complex in a mannersimilar to IL-15. Such binding to the IL-15 receptor complex can bemeasured using suitable techniques such as flow cytometry which areknown to the skilled artisan.

Preferably, the IL-15 is directly utilized by the mammalian cell or cellline expressing it and is not secreted to the culture supernatant inamounts supporting the growth and survival of bystander cells which donot express IL-15 themselves.

—Selectable Marker for Gene Transfer

A marker gene is a suitable means in molecular biology for determiningwhether the transfer of specific nucleic acid(s) (such as DNA, hereingene of interest or effector gene) into a host cell has been successful.There are two types of marker genes: selectable markers and markers forscreening.

A “selectable marker” or “selection marker” will either protect the hostcell from a selective agent that would normally kill it or prevent itsgrowth or is required for the host cells growth and survival. It is agene introduced into the host cell that confers a trait suitable forartificial selection.

In most applications, only one in several hundred cells will take up thespecific nucleic acid(s) (such as DNA encoding a gene/protein ofinterest). Rather than checking every single cell, a selective agent isused to kill all cells that do not contain the foreign nucleic acid(s)or only allows cells containing the foreign nucleic acid(s) to grow,thus, leaving only the desired ones. As discussed above, selectablemarkers are often antibiotic resistance genes or bacterial enzymes.

Thus, a “selectable marker” is a gene whose expression allows one toidentify and selectively enrich cells that have been transformed,transfected or transduced with a nucleic acid construct containing themarker gene.

The term “gene transfer” refers to the introduction of a nucleic acid(construct) (expression or gene construct) of interest into themammalian cell or cell line by any way, such as transformation,transfection, microinjection, particle-mediated transfer, transductionwith a viral vector. These techniques are known to the skilled artisan.

According to the invention, IL-15 is used as selectable marker for exvivo or in vitro gene transfer.

According to the invention, IL-15 is used as selectable marker for thegene transfer of another nucleic acid or gene, which is transferred intothe host cell at the same time (i.e. together with the IL-15).

Said other nucleic acid or gene is a “gene of interest” or “effectorgene” (wherein these terms are used interchangeably throughout thisspecification) which encodes a “protein of interest” or “effectorprotein” (wherein these terms are used interchangeably throughout thisspecification).

Preferably, the gene transfer into the mammalian cell or cell line, forwhich IL-15 is used as the selectable marker, is the transfer of IL-15together with at least one gene of interest (other than IL-15) encodinga protein of interest (other than IL-15) into the mammalian cell or cellline.

Thereby, the nucleic acid/coding sequence of IL-15 and the at least onegene of interest are transferred into the cell using one/the sameexpression or gene construct or using different expression or geneconstructs.

In other embodiments of the invention, more than one gene of interesteach encoding a protein of interest is/are transferred into themammalian cell or cell line, such as two, three, four or more genes ofinterest.

The at least one gene of interest preferably encodes a protein oftherapeutic interest, preferably a chimeric antigenic receptor (CAR).

—Host Cells

Preferably, the mammalian cell or cell line is a human cell or cellline.

The mammalian cell or cell line, in particular the human cell or cellline, requires cytokines for growth and survival when it is notmodified, i.e. not expressing IL-15. Said cytokines are preferably oneor more cytokine that bind to a receptor complex that contains thecommon gamma chain of the IL-2 receptor and include but are not limitedto the cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, morepreferably IL-2.

In particular, the mammalian cell or cell line, in particular the humancell or cell line, is in non-modified state preferably dependent on saidcytokine(s) for its growth and survival and is unable to produce anysignificant amounts of IL-15, but in modified state it produces IL-15 inan amount sufficient to sustain growth and survival without the need ofsaid cytokine(s). In in vitro culture, the mammalian cell or cell line,in particular the human cell or cell line, requires for growth andsurvival in non-modified state that above cytokines are addedexogenously.

According to the invention, the mammalian cell or cell line is selectedfrom effector cells of the immune system, such as lymphocytes includingbut not limited to cytotoxic lymphocytes, T cells, cytotoxic T cells, Thelper cells, Th17 T cells, natural killer (NK) cells, natural killer T(NKT) cells, mast cells, dendritic cells, killer dendritic cells, Bcells.

According to the invention, the mammalian cell or cell line is selectedfrom effector cells of the immune system which require cytokines(preferably one or more cytokine that bind to a receptor complex thatcontains the common gamma chain of the IL-2 receptor and include but arenot limited to the cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21,more preferably IL-2) for growth and survival.

More preferably, the human cell or cell line is selected from naturalkiller (NK) cells and T cells (such as cytotoxic T lymphocytes (CTLs)).

Due to the endogenous expression of IL-15 as selectable marker gene, themammalian cells of the invention do not require, in in vitro culture,any exogenously added cytokines (preferably one or more cytokine thatbind to a receptor complex that contains the common gamma chain of theIL-2 receptor and include but are not limited to the cytokines IL-2,IL-4, IL-7, IL-9, IL-15 and IL-21, more preferably IL-2) for growth andsurvival, which allows the selection of these cells by withdrawal of theexogenous cytokines, which e.g. has advantages for the use of thesecells in in vivo applications.

Thus, the present invention provides the use of interleukin-15 (IL-15)as selectable marker for ex vivo or in vitro gene transfer into amammalian cell or cell line,

wherein the mammalian cell or cell line is selected from effector cellsof the immune system which require cytokines for growth and survival,

wherein said cytokines include but are not limited to IL-2, IL-4, IL-7,IL-9, IL-15 and IL-21,

wherein upon gene transfer of the IL-15 into said mammalian cell or cellline the expression of the IL-15 as selectable marker results insurvival or growth of the mammalian cell or cell line in the absence ofsaid cytokines,

wherein the IL-15 has the amino acid sequence of SEQ ID NO. 1 or anamino acid sequence that has at least 95% sequence identity to SEQ IDNO. 1,

or is encoded by the nucleotide sequence of SEQ ID NO. 2 or a nucleotidesequence with at least 95% sequence identity to SEQ ID NO. 2.

Expression of chimeric antigen receptors (CAR) with defined target-cellspecificity in lymphocytes results in genetically modified variants ofthe lymphocytes that selectively target and eliminate defined targetcells including but not limited to malignant cells carrying a respectivetumor-associated surface antigen or virus infected cells carrying avirus-specific surface antigen (Uherek et al., 2001). Therebygamma-retroviral or lentiviral vectors are commonly employed for genetransfer into cytotoxic lymphocytes, but depending on the particularcell type used this can result in only a small proportion of cells beingtransduced successfully. Object matter of the invention are methods andmeans such as gene constructs for expression in lymphocytes that carrycDNA of human interleukin-15 (IL-15) as a selectable marker gene inaddition to another gene of interest such as a CAR or another(other)gene(s) of interest with therapeutic value.

As discussed above, depending on the cell type used, the relatively lowtransduction efficiency of viral vectors employed for geneticmodification of lymphocytes, in particular NK cells, with effector genesof therapeutic value such as genes encoding CAR limits the relativeproportion of gene-modified cells in the transduced cell pool. Inprinciple, inclusion of a selectable marker gene in the vectorconstructs would allow selection and enrichment of gene-modified cellsprior to potential therapeutic applications in adoptive immunotherapy.However, available selection markers such as bacterial resistance genesand bacterial enzymes cannot be used due to their non-human origin andtheir potential immunogenicity. Furthermore, selection using suchmarkers requires antibiotics or toxic reagents which must be added tothe culture medium. Therefore the aim of the technical solutiondescribed herein was the generation of methods and means such as vectorsfor gene transfer into lymphocytes that contain a selectable marker geneof human origin which allows selective enrichment of gene-modified cellsin standard culture medium without addition of toxic compounds.Cytotoxic lymphocytes such as NK cells or CTL require (exogenous)cytokines (e.g. IL-2) for growth and survival, which can at the sametime be substituted with the related cytokine IL-15 used as theselectable marker.

Object matter of the invention are gene constructs for expression inlymphocytes that carry cDNA of human interleukin-15 (IL-15) as aselectable marker gene in addition to another gene of interest such as aCAR or another effector gene of therapeutic value. After gene transferinto cytokine-dependent lymphocytes like NK cells or T lymphocytes, onlycells that have successfully incorporated the transferred construct areable to grow in the absence of exogenously added cytokines and can suchbe selected for simply by withdrawal of exogenous cytokines. At the sametime this autocrine production of IL-15 provides the gene-modifiedlymphocytes with a growth and survival signal indispensable for theircontinued functional activity.

The inventors have now demonstrated the suitability or use of IL-15 asselectable marker gene. Our technical solution is not based on IL-2 buton the use of IL-15. Thereby, using IL-15 circumvents potential sideeffects of IL-2 in vivo, since IL-15 does not support the suppressingactivity of regulatory T cells (Wuest et al., 2008). In contrast toZhang et al., who previously described expression of IL-15 in NK cells(Zhang et al., 2004), our technical solution allows only growth of NKcells transduced with constructs (vectors) encoding IL-15 in thecomplete absence of exogenous cytokines, which is a requirement for theuse of IL-15 as a selection marker. NK cells expressing such constructsaccording to the invention do not secrete measurable amounts of IL-15into the culture supernatant but utilize all IL-15 producedendogenously. Hence, IL-15 expressing NK cells do not support the growthor survival of IL-15 negative bystander cells making IL-15 suitable asselectable marker gene (as has been demonstrated in e.g. FIG. 6)

According to the invention, the expression of the IL-15 does not resultin the secretion of IL-15 into the culture supernatant in amountssufficient to support survival and growth of cells that are nottransformed or transduced with the IL-15 and/or are not expressing theIL-15 themselves.

—Embodiment Wherein the Protein of Therapeutic Interest is a CAR

In one embodiment of the invention the protein of therapeutic interestis a chimeric antigen receptor (CAR).

A CAR comprises

-   -   (i) a signal peptide;    -   (ii) a target specific recognition domain, binding an antigen,        receptor, peptide ligand or protein ligand of the target,        wherein the target is a cell or a virus;    -   (iii) a linker region, connecting domain (ii) and domain (iv);        and    -   (iv) an effector domain comprising a transmembrane region and        one or more intracellular signaling domains.

A “chimeric antigen receptor” is a cell surface receptor protein and,thus, comprises an extracellular portion (domains (i) and (ii) and(iii)), a transmembrane portion (contributed by/comprised in domain(iv)) and a cytoplasmic portion (contributed by/comprised in domain(iv)), and can thus be inserted into the plasma membrane of the hostcell. The functionality of a CAR within a host cell is detectable in anassay suitable for demonstrating the signaling potential of said proteinupon binding of a particular ligand. Such assays are available to theskilled artisan. Upon binding to the target, CARs link to endogenoussignaling pathways in a cell (an effector cell) and generate certainactivating signals (depending on the effector domain).

The target specific recognition domain (ii) binds an antigen, receptor,peptide ligand or protein ligand of the target.

The target specific recognition domain (ii) preferably comprises

-   -   an antigen binding domain derived from an antibody against an        antigen of the target, or    -   a peptide binding an antigen of the target, or    -   a peptide or protein binding an antibody that binds an antigen        of the target, or    -   a peptide or protein ligand (including but not limited to a        growth factor, a cytokine or a hormone) binding a receptor on        the target, or    -   a domain derived from a receptor (including but not limited to a        growth factor receptor, a cytokine receptor or a hormone        receptor) binding a peptide or protein ligand on the target.

Preferably, the target is a cell or a virus.

The target specific recognition domain serves for the targeting of theCAR or a respective cell expressing/carrying the CAR on its surface to aspecific target. Binding of the target specific recognition domain ofthe CAR to its cognate target on the surface of target cells/virusesfurthermore transmits a signal into the CAR-expressing immune effectorcells via the intracellular signaling domain(s) of the CAR whichactivates the endogenous cytotoxic activity of such imune effectorcells.

Where domain (ii) of the CAR binds an antigen of the target, examples ofthe antigen are

-   -   a tumor-associated surface antigen (such as ErbB2 (HER2/neu),        carcinoembryonic antigen (CEA), epithelial cell adhesion        molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR        variant III (EGFRvIII), CD19, CD₂O, CD30, CD40,        disialoganglioside GD2, a major histocompatibility complex (MHC)        molecule presenting a tumor-specific peptide epitope);    -   a lineage-specific or tissue-specific surface antigen (such as        CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4,        B7-1 (CD80), B7-2 (CD86), endoglin, a major histocompatibility        complex (MHC) molecule) or    -   a virus-specific surface antigen (such as an HIV-specific        antigen (e.g. HIV gp120), an EBV-specific antigen, a        CMV-specific antigen, a HPV-specific antigen, a HBV-specific        antigen, a HCV-specific antigen, a Lassa Virus-specific antigen,        an Influenza Virus-specific antigen).

Where domain (ii) of the CAR comprises an antigen binding domain, theantigen binding domain is preferably derived from an antibody or anantibody fragment, such as a single chain Fv (scFv) fragment, a Fabfragment, a diabody, a variable domain of the antibody heavy chain orantibody light chain.

More preferably, the domain (ii) of the CAR binds an antigen of thetarget and the antigen is a tumor-associated surface antigen, such asEpCAM or ErbB2.

The linker region (iii) of the CAR connects the target specificrecognition domain (ii) and the effector domain (iv). The linker regionserves as a flexible spacer between the target specific recognitiondomain (ii) and the effector domain (iv). It ensures the necessaryaccessibility and flexibility of the target specific recognition domain(ii). The linker region is understood to be essential for thefunctionality of the CARs.

CARs typically contain a linker region derived from the alpha-chain ofthe human CD8 molecule which provides a flexible connection betweencell-targeting and signaling/effector domains (Uherek et al., 2002;Müller et al., 2008).

In one embodiment, the linker region (iii) of the CAR comprises a hingeregion derived from the human CD8 alpha-chain, wherein said human CD8alpha-chain hinge region has preferably the amino acid sequence of SEQID NO. 3, or an amino acid sequence that has at least 80% sequenceidentity to the amino acid sequence of SEQ ID NO. 3, preferably at least90% sequence identity, more preferably at least 95% sequence identity or99% sequence identity to the amino acid sequence of SEQ ID NO. 3, or anamino acid sequence that differs in one, two, three, four or more, up totwelve, amino acid residues from the amino acid sequence of SEQ ID NO. 3(i.e. differs in one, two, three, four, five, six, seven, eight, nine,ten, eleven or twelve), wherein “differ” refers toreplacement/substitution, addition or deletion, such as conservativesubstitution(s) of amino acid residues.

SEQ ID NO. 3: ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD

The effector domain (iv) of the CAR comprises a transmembrane region andone or more intracellular signaling domains. The effector domain servesthe coupling of the target/antigen recognition to the intracellularsignaling machinery. Binding of the target specific recognition domain(ii) of the CAR to its cognate target on the surface of targetcells/viruses furthermore transmits a signal into the CAR-expressingimmune effector cells via the intracellular signaling domain(s) of theCAR (which are part of the effector domain) which activates theendogenous cytotoxic activity of such immune effector cells.

In an embodiment, the effector domain (iv) comprises or consists of (is)the zeta-chain of the human CD3 complex of the T-cell receptor or afragment thereof or a functional equivalent thereof or a fusion with afurther protein (or fragment thereof), such as a fragment of the humancostimulatory CD28 receptor.

In an embodiment, the zeta-chain of the human CD3 complex of the T-cellreceptor has the amino acid sequence of SEQ ID NO. 4.

A “functional equivalent” has less sequence identity (such as at least80% sequence identity, preferably at least 90% sequence identity, morepreferably at least 95% sequence identity or 99% sequence identity) butis a functional zeta-chain of the CD3 complex of the T-cell receptor.According to the invention, the zeta chain is of human origin. Withinthe TCR the CD3 zeta chain exists as a disulfide homodimer. A“functional CD3 zeta chain” or “a functional zeta-chain of the CD3complex of the T-cell receptor” is a protein which upon expression in Tcell hybridomas deficient in endogenous zeta expression is capable ofrestoring in said hybridomas a functionally active TCR.

Generally, a person skilled in the art is aware of the fact that someamino acid exchanges in the amino acid sequence of a protein or peptidedo not have any influence on the (secondary or tertiary) structure,function and activity of the protein or peptide (at all). Amino acidsequences with such “neutral” amino acid exchanges as compared to theamino acid sequences disclosed herein fall within the scope of thepresent invention.

Transgenic Cells Expressing IL-15 and a Gene of Interest

As described above, the present invention provides transgenic mammaliancells or cell lines expressing IL-15 as selectable marker andco-expressing at least one protein of interest (other than IL-15)encoded by at least one gene of interest (other than IL-15).

Preferably, the transgenic mammalian cell or cell line is a human cellor cell line.

The mammalian cell or cell line, in particular the human cell or cellline, requires cytokines for growth and survival when it is notmodified, i.e. not expressing IL-15. Said cytokines are preferably oneor more cytokine that bind to a receptor complex that contains thecommon gamma chain of the IL-2 receptor and include but are not limitedto the cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, morepreferably IL-2.

In particular, the mammalian cell or cell line, in particular the humancell or cell line, is in non-modified state preferably dependent on saidcytokine(s) for its growth and survival and is unable to produce anysignificant amounts of IL-15, but in modified state it produces IL-15 inan amount sufficient to sustain growth and survival without the need ofsaid cytokine(s). In in vitro culture, the mammalian cell or cell line,in particular the human cell or cell line, requires for growth andsurvival in non-modified state that above cytokines are addedexogenously.

According to the invention, the transgenic mammalian cell or cell lineis selected from effector cells of the immune system, such aslymphocytes including but not limited to cytotoxic lymphocytes, T cells,cytotoxic T cells, T helper cells, Th17 T cells, natural killer (NK)cells, natural killer T (NKT) cells, mast cells, dendritic cells, killerdendritic cells, B cells.

According to the invention, the transgenic mammalian cell or cell lineis selected from effector cells of the immune system which requirecytokines (preferably one or more cytokine that bind to a receptorcomplex that contains the common gamma chain of the IL-2 receptor andinclude but are not limited to the cytokines IL-2, IL-4, IL-7, IL-9,IL-15 and IL-21, more preferably IL-2) for growth and survival.

Preferably, the human cell or cell line is selected from natural killer(NK) cells and T cells (such as cytotoxic T lymphocytes (CTLs)).

Due to the endogenous expression of IL-15 as selectable marker gene, themammalian cells of the invention do not require, in in vitro culture,any exogenously added cytokines (preferably one or more cytokine thatbind to a receptor complex that contains the common gamma chain of theIL-2 receptor and include but are not limited to the cytokines IL-2,IL-4, IL-7, IL-9, IL-15 and IL-21, more preferably IL-2) for growth andsurvival, which allows the selection of these cells by withdrawal of theexogenous cytokines, which e.g. has advantages for the use of thesecells in in vivo applications.

The present invention provides a transgenic mammalian cell or cell lineexpressing IL-15, as defined herein, as selectable marker for genetransfer and co-expressing at least one protein of interest encoded byat least one gene of interest,

wherein the mammalian cell or cell line is selected from effector cellsof the immune system which require cytokines for growth and survival,

wherein said cytokines include but are not limited to IL-2, IL-4, IL-7,IL-9, IL-15 and IL-21,

wherein the protein of interest is a protein other than IL-15 and thegene of interest is a gene other than IL-15,

and wherein (upon gene transfer of the IL-15 and the gene/protein ofinterest into said mammalian cell or cell line) the expression of theIL-15 as selectable marker results in survival or growth of themammalian cell or cell line in the absence of said cytokines.

Preferably (and as has been discussed herein), the expression of theIL-15 does not result in the secretion of IL-15 into the culturesupernatant in amounts sufficient to support survival and growth ofcells that are not transformed or transduced with the IL-15 and/or arenot expressing the IL-15 themselves.

The IL-15 (gene) and the at least one gene of interest can betransferred into the transgenic cell of the invention using:

-   -   (a) one expression or gene construct    -   which comprises both a nucleotide sequence encoding (human)        IL-15, i.e. a coding sequence of (human) IL-15, and the gene of        interest, or    -   (b) different expression or gene constructs (at least two)    -   wherein one construct comprises a nucleotide sequence encoding        (human) IL-15, i.e. a coding sequence of (human) IL-15, and the        other construct(s) comprise(s) the gene(s) of interest.

An “expression or gene construct” (wherein both terms are usedinterchangeably throughout this specification) refers to a nucleic acidconstruct, usually an expression vector or plasmid, that is used tointroduce a specific gene sequence into a target cell. Once theexpression or gene construct is inside the cell, the protein that isencoded by the gene is produced by the cellular transcription andtranslation machinery. The expression or gene construct is designed tocontain respective regulatory sequences that act as enhancer andpromoter regions and lead to efficient transcription of the gene carriedon the construct, including promoter and terminator sequences). The goalof a well-designed expression or gene construct is the production oflarge amounts of stable mRNA, and therefore proteins.

In an embodiment of the invention utilizing one expression construct(a), the coding sequence of (human) IL-15 and the gene of interest areseparated by a regulatory element, preferably an internal ribosome entrysite (IRES), enabling their simultaneous expression under the control ofa single promoter.

The expression or gene construct comprising the IL-15 (gene) and/or theat least one gene of interest is preferably a (DNA) plasmid or a viralvector, such as a lentiviral vector, a gamma-retroviral vector or anadeno-associated virus vector.

The nucleic acids of this invention comprise DNA (such as dsDNA, ssDNA,cDNA), RNA (such as dsRNA, ssRNA, mRNA), combinations thereof orderivatives (such as PNA) thereof.

The expression or gene construct comprises a nucleotide sequenceencoding (human) IL-15, i.e. a coding sequence of (human) IL-15, asdefined herein above.

Suitable expression or gene constructs and plasmids are known to theskilled artisan, such as (DNA) plasmids or viral vectors (e.g.lentiviral vector, a gamma-retroviral vector or an adeno-associatedvirus vector).

In other embodiments of the invention, more than one gene of interesteach encoding an protein of interest is transferred into the mammaliancell or cell line, such as two, three, four or more genes of interest.

Preferably, the at least one gene of interest encodes a protein oftherapeutic interest.

In an embodiment, the protein of therapeutic interest is a chimericantigenic receptor (CAR), wherein the CAR is as defined herein above.

The present invention also encompasses a method of producing transgenic(gene-modified) mammalian cells or cell lines expressing IL-15 asselectable marker and co-expressing at least one protein of interestencoded by at least one gene of interest, wherein the method comprises:

-   -   selection and construction of expression or gene construct(s)        comprising either (a—one construct) both a nucleotide sequence        encoding (human) IL-15, i.e. a coding sequence of (human) IL-15,        and the gene of interest, or (b—different constructs) comprising        a nucleotide sequence encoding (human) IL-15, i.e. a coding        sequence of (human) IL-15, or the gene(s) of interest;    -   genetic modification of the cells by transfer of the expression        or gene construct(s) into the cell;    -   selection of the transgenic (gene-modified) cells.        Medical Uses of the Transgenic Cells

As described above, the present invention provides the transgenicmammalian cells or cell lines of the invention (which express IL-15 asselectable marker and co-express at least one protein of interestencoded by at least one gene of interest) for use as a medicament.

As described above, the present invention provides the transgenicmammalian cells or cell lines of the invention (which express IL-15 asselectable marker and co-express at least one protein of interestencoded by at least one gene of interest) for use in the treatment ofcancer or in immunotherapy, preferably in adoptive, target-cell specificimmunotherapy.

“Adoptive, target-cell specific immunotherapy” refers to a form oftherapy in which immune cells are transferred to tumor-bearing hosts.The immune cells have antitumor reactivity and can mediate direct orindirect antitumor effects.

“Adoptive, target-cell specific immunotherapy” or “adoptive cell therapy(ACT)” is a treatment that uses immune effector cells, such aslymphocytes with anti-tumour activity, expanded in vitro and infusedinto the patient with cancer. ACT using autologous tumour-infiltratinglymphocytes has emerged as the most effective treatment for patientswith metastatic melanoma and can mediate objective cancer regression inapproximately 50% of patients. The use of donor lymphocytes for ACT isan effective treatment for immunosuppressed patients who developpost-transplant lymphomas (reviewed in Rosenberg et al., 2008). However,the ability to genetically engineer human lymphocytes and use them tomediate cancer regression in patients, which has recently beendemonstrated (see Morgan et al, 2006), has opened possibilities for theextension of ACT immunotherapy to patients with a wide variety of cancertypes and is a promising new approach to cancer treatment. Thus,lymphocytes genetically engineered with chimeric antigen receptors(CAR), such as provided by this invention, are very suitable for ACT andopen more possibilities in the treatment of cancer. Especially, sincestudies have clearly demonstrated that the administration of highly avidanti-tumour T cells directed against a suitable target can mediate theregression of large, vascularized, metastatic cancers in humans andprovide guiding principles as well as encouragement for the furtherdevelopment of immunotherapy for the treatment of patients with cancer.

The mammalian cells and cell lines of the invention, in particular thehuman immune effector cells, are very suitable for medical applications,in particular for ACT, because:

-   -   they carry a selectable marker of human origin (thus not        eliciting an immune response);    -   they require no exogenous cytokines for growth and survival;    -   they show highly functional CAR-mediated cytotoxicity in absence        of exogenous cytokines (in the embodiment with a CAR as a        protein/gene of interest);    -   they do not support the growth of IL-15 negative bystander        cells, because the effect of the IL-15 is limited to the cell        producing it;    -   they utilize the IL-15 produced endogenously and do not secrete        measurable amounts of IL-15 into the culture supernatant;    -   they are designed for the coexpression of at least one gene of        therapeutic interest.        Treatment Methods

Furthermore, the present invention provides methods for the treatment ofdiseases, in particular cancer, and methods of immunotherapy, preferablyincluding adoptive, target-cell specific immunotherapy.

The method for the treatment of diseases, in particular cancer,according to the present invention comprises

-   -   administering to a subject in a therapeutically effective amount    -   (a) a transgenic mammalian cell or cell line as obtained and        defined herein above; and    -   (b) optionally, respective excipient(s).

The method of immunotherapy, preferably including or utilizing adoptive,target-cell specific immunotherapy, according to the present inventioncomprises

-   -   administering to a subject in a therapeutically effective amount    -   (a) a transgenic mammalian cell or cell line as obtained and        defined herein above; and    -   (b) optionally, respective excipient(s).

A “therapeutically effective amount” of a transgenic mammalian cell orcell line of this invention refers to the amount that is sufficient totreat the respective disease or achieve the respective outcome of theadoptive, target-cell specific immunotherapy.

The following examples and drawings illustrate the present inventionwithout, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic representation of expression constructs.

-   (A) Lentiviral transfer vector with IL-15 cDNA inserted as a    selectable marker downstream of an internal ribosome entry site    (IRES). The vector also contains a SacII cloning site for the    insertion of a gene of interest. Expression is driven by the Spleen    Focus Forming Virus promoter (SFFV).-   (B) Lentiviral transfer vector encoding IL-15 under the control of    the SFFV promoter and followed by an IRES sequence and cDNA encoding    enhanced green fluorescent protein (EGFP) as a marker.-   (C) Lentiviral transfer vector encoding a chimeric antigen receptor    (CAR) as a gene of interest under the control of the SFFV promoter,    followed by an IRES sequence and IL-15 cDNA as a selectable marker.    The CAR is composed of immunoglobulin heavy chain signal peptide    (SP), a single-chain Fv antibody fragment specific for a target cell    surface antigen (scFv), a CD8 alpha-chain hinge region as a flexible    linker (CD8 alpha), and CD3 zeta-chain or a composite CD28-CD3    zeta-chain fusion as a signaling domain (zeta).

FIG. 2 Selective enrichment of NK Cells expressing IL-15 as a selectablemarker.

NK cells were transduced with a lentiviral vector encoding IL-15 as aselectable marker followed by an internal ribosome entry site andenhanced green fluorescent protein (EGFP) as a gene of interest(NK/IL-15 EGFP; see FIG. 1B). EGFP expression of cells was analyzed byflow cytometry at different time points after transduction. Maintenanceof the pool of transduced NK cells in regular growth medium containing100 IU/mL IL-2 did not result in selective enrichment of EGFP expressingcells (upper right panel) when compared to untransduced NK cells (leftpanels). Maintenance of the pool of transduced NK cells in growth mediumwithout IL-2 for 14 days resulted in selective enrichment ofsuccessfully transduced NK cells co-expressing IL-15 and EGFP (lowerright panel).

FIG. 3 Growth of IL-15 expressing NK cells in the absence of exogenouscytokines.

NK cells were transduced with a lentiviral vector encoding IL-15 as aselectable marker followed by an internal ribosome entry site andenhanced green fluorescent protein (EGFP) as a gene of interest (seeFIG. 1B). Transduced NK cells (NK/IL-15 EGFP) or untransduced NK cellsas controls were either grown in regular growth medium containing 100IU/mL IL-2 (+IL-2), or in the same medium lacking exogenous cytokines(no IL-2) as indicated. At different time points cell growth wasanalyzed in MTT cell viability assays. While no significant growth ofuntransduced NK cells was observed in the absence of exogenous cytokines(open circles), NK cells expressing IL-15 as a selectable markercontinued to grow in the absence of IL-2 (open boxes) and displayedgrowth kinetics similar to control NK cells grown in the presence ofIL-2 (filled circles).

FIG. 4. Cytotoxic activity of NK cells expressing CAR and IL-15.

NK cells were transduced with a lentiviral vector encoding anEpCAM-specific chimeric antigen receptor (CAR) followed by an internalribosome entry site and IL-15 as a selectable marker (NK/CAR IL-15; seeFIG. 1C). Control cells were transduced with a lentiviral vector onlyencoding IL-15 downstream of an IRES sequence (NK/IRES-IL-15; see FIG.1A). Gene-modified, IL-15 expressing cells were selected by withdrawalof exogenous IL-2 from the culture medium.

-   (A) Analysis of CAR surface expression. Expression of CAR on the    surface of NK/CAR IL-15 cells was investigated by FACS analysis    using an antibody detecting a sequence tag included in the    EpCAM-specific CAR (dark gray). NK cells transduced with IRES-IL-15    vector served as control (light gray).-   (B)-(D) NK cells co-expressing CAR and IL-15 (NK/CAR IL-15) or NK    cells only expressing IL-15 (NK/IRES-IL-15) were co-cultured in the    absence of exogenous cytokines at different effector to target (E:T)    ratios with NK-sensitive K562 erythroleukemic control cells (B),    EpCAM-expressing MDA-MB468 breast carcinoma cells (C), or    EpCAM-negative MDA-MB435 melanoma cells (D). As shown in (C), NK    cells expressing IL-15 and the EpCAM-specific CAR showed    EpCAM-specific and highly effective cell killing (open bars) when    compared to NK cells expressing IL-15 but no CAR (filled bars).

FIG. 5 Growth of IL-15 expressing cytotoxic T lymphocytes in the absenceof exogenous cytokines.

Cytotoxic T lymphocytes (CTL) were transduced with a lentiviral vectorencoding IL-15 as a selectable marker followed by an internal ribosomeentry site and enhanced green fluorescent protein (EGFP) as a gene ofinterest (see FIG. 1B). Transduced CTL (CTL/IL-15 EGFP) or untransducedCTL as controls were either grown in regular growth medium containing 50IU/mL IL-2 (+IL-2), or in the same medium lacking exogenous cytokines(no IL-2) as indicated. At different time points cell growth wasanalyzed in MTT cell viability assays. While no significant growth ofuntransduced CTL was observed in the absence of exogenous cytokines(open circles), CTL expressing IL-15 as a selectable marker continued togrow in the absence of IL-2 (open boxes) and displayed growth kineticssimilar to control CTL grown in the presence of IL-2 (filled circles)and CTL expressing IL-15 grown in the presence of IL-2 (filled boxes).

FIG. 6 1L-15 bioactivity in culture supernatant of IL-15 expressing NKcells.

NK cells were transduced with a lentiviral vector encoding anEpCAM-specific CAR followed by an internal ribosome entry site and IL-15as a selectable marker, and gene-modified, IL-15 expressing cells wereselected by withdrawal of exogenous IL-2 from the culture medium asdescribed in the legend for FIG. 4. Conditioned culture medium wascollected from CAR and IL-15 expressing NK cells grown in the absence ofIL-2 (NK/CAR/IL-15), and as a control from untransduced NK cells thatwere left in medium without IL-2 (NK). Then the growth ofIL-2/IL-15-dependent murine CTLL-2 cells in the presence of theconditioned culture medium was analyzed at different time points in MTTcell viability assays in comparison to growth of CTLL-2 cells in regulargrowth medium containing 50 IU/mL IL-2 (+IL-2) or regular growth mediumlacking IL-2 (no IL-2). While CTLL-2 indicator cells continued to growin regular growth medium containing exogenous IL-2 (filled circles),conditioned medium from CAR and IL-15 expressing NK cells (filled boxes)and untransduced NK cells (open circles), and regular growth mediumlacking exogenous IL-2 (open boxes) did not support growth of CTLL-2cells.

EXAMPLES Example 1

Generation of IL-15 expression constructs. Different lentiviral vectorsbased on SIEW were used to analyze the suitability of interleukin-15(IL-15) as a selectable marker for enrichment of gene-modifiedlymphocytes. In all vectors, expression of IL-15 and additional genes isdriven by a spleen focus forming virus promoter (SFFV). For expressionof IL-15 as a single gene of interest, cDNA encoding human IL-15 wasinserted downstream of an internal ribosome entry site (IRES) inlentiviral vector SIEW (FIG. 1A). To analyze functionality of IL-15 as aselectable marker and IL-15 mediated selection of cells co-expressingIL-15 and enhanced green fluorescent protein (EGFP) as a model gene ofinterest, IL-15 cDNA was inserted upstream of an IRES sequence and cDNAencoding EGFP in lentiviral vector SIEW (FIG. 1B). To analyzefunctionality of IL-15 as a selectable marker and IL-15 mediatedselection of cells co-expressing IL-15 and a chimeric antigen receptor(CAR) as a gene of interest with therapeutic activity, a bicistronicvector was generated that encodes a CAR, followed by an IRES sequenceand IL-15 cDNA (FIG. 1C). The CAR is composed of an immunoglobulin heavychain signal peptide, a single-chain Fv antibody fragment specific for atarget cell surface antigen on tumor cells, a CD8 alpha-chain hingeregion as a flexible linker, and CD3 zeta-chain as a signaling domain.

Transduction of NK Cells and CTL. VSV-G pseudotyped lentiviral vectorparticles were produced by transient triple transfection of 293T cellswith the transfer vector together with the packaging constructs pMD-VSVGand 8.91. Lentiviral vector was used for transduction of NK cells andCTL, and successfully transduced NK cells and CTL were selected by IL-2withdrawal starting two days after transduction.

Selective enrichment of NK cells expressing IL-15 as a selectablemarker. The functionality of IL-15 as a selectable marker for enrichmentof gene-modified lymphocytes was tested by transduction of NK cells witha lentiviral vector encoding IL-15 followed by an internal ribosomeentry site and enhanced green fluorescent protein (EGFP) as a gene ofinterest. The transduction rate in this experiment was approximately 2%indicated by the proportion of EGFP-positive cells, which remained atthis level upon culture in medium containing exogenous IL-2 (FIG. 2). Incontrast, culture of transduced cells in selection medium lackingexogenous cytokines for 14 days resulted in selective enrichment ofgene-modified cells indicated by a marked increase of EGFP-positivecells to approximately 97% (FIG. 2). These results demonstrate thatIL-15 is effective as a selectable marker gene in a bicistronic vector,allowing enrichment of gene-modified lymphocytes expressing IL-15together with another gene of interest. Furthermore, IL-15 is sufficientto support long-term growth and survival of the selected lymphocytes inthe absence of exogenous cytokines.

Growth of IL-15 expressing NK cells and CTL in the absence of exogenouscytokines. Proliferation of NK cells and CTL expressing IL-15 as aselectable marker and EGFP as a gene of interest was analyzed in MTTcell viability assays. Untransduced NK cells and CTL served as control.While no significant growth of untransduced NK cells and CTL wasobserved in the absence of exogenous cytokines, NK cells (FIG. 3) andCTL (FIG. 5) expressing IL-15 as a selectable marker continued to growin the absence of IL-2 and displayed growth kinetics similar to controlNK cells and CTL grown in the presence of IL-2. These resultsdemonstrate that expression of IL-15 as a selectable marker issufficient to support long-term growth and survival of cytotoxiclymphocytes in the absence of exogenous cytokines.

Expression of chimeric antigen receptor and cytotoxic activity of NKcells selected using IL-15 as a selectable marker. Expression andfunctionality of chimeric antigen receptors in NK cells transduced witha lentiviral vector encoding a CAR as a gene of interest and IL-15 as aselectable marker was tested by flow cytometry and in FACS-basedcytotoxicity assays. NK cells were transduced with a lentiviral vectorencoding an EpCAM-specific chimeric antigen receptor (CAR) followed byan internal ribosome entry site and IL-15 as a selectable marker.Gene-modified, IL-15 expressing cells were selected by withdrawal ofexogenous IL-2 from the culture medium. Expression of CAR on the surfaceof selected cells was investigated by FACS analysis using an antibodydetecting a sequence tag included in the EpCAM-specific CAR. It wasfound that selection for IL-15 expressing cells by IL-2 withdrawalresulted in a surviving cell population homogeneously expressing CAR onthe cell surface (FIG. 4A). Functionality of these cells was tested incytotoxicity assays without addition of exogenous cytokines. Thereby NKcells co-expressing EpCAM-specific CAR and IL-15, and NK cells onlyexpressing IL-15 displayed similar cytotoxic activity towardsNK-sensitive K562 erythroleukemic control cells, but only littleactivity against NK-resistant and EpCAM-negative MDA-MB435 melanomacells (FIG. 4B, D). When cytotoxic activity towards EpCAM-positiveMDA-MB468 breast carcinoma cells was tested, NK cells co-expressing theEpCAM-specific CAR and IL-15 showed EpCAM-specific and highly effectivecell killing, while control cells only expressing IL-15 did not (FIG.4C). These results demonstrate that IL-15 is effective as a selectablemarker gene in a bicistronic vector, allowing enrichment of cytotoxiclymphocytes expressing a CAR as a gene of interest. Furthermore, IL-15facilitates full functionality of the selected lymphocytes in theabsence of exogenous cytokines.

IL-15 bioactivity in culture supernatant of IL-15 expressing NK cells.To test whether IL-15 is secreted by IL-15 expressing NK cells inamounts sufficient to support survival and growth of non-transducedbystander cells, IL-15 bioactivity in conditioned culture supernatant ofIL-15 expressing NK cells was investigated. NK cells were transducedwith a lentiviral vector encoding an EpCAM-specific CAR followed by aninternal ribosome entry site and IL-15 as a selectable marker asdescribed above. Gene-modified, IL-15 expressing cells were selected bywithdrawal of exogenous IL-2 from the culture medium. Conditioned mediumfrom the gene-modified NK cells growing in the absence of IL-2 wascollected after three days of culture, and the ability of untransducedIL-2/IL-15-dependent CTLL-2 cells to grow in the presence of conditionedNK cell medium was analyzed in MTT cell viability assays. Conditionedmedium from untransduced NK cells served as control. While significantgrowth of CTLL-2 cells was observed in regular growth medium containingIL-2, conditioned medium from CARand IL-15 expressing NK cells likeconditioned medium from untransduced NK cells and growth medium lackingIL-2 did not support growth of CTLL-2 cells (FIG. 6). These resultsdemonstrate that IL-15 expressing cytotoxic lymphocytes, whilesupporting their own growth via ectopic production of IL-15 (see FIGS.2, 3, 5), do not secrete IL-15 in amounts high enough to also supportgrowth and survival of untransduced bystander cells. Hence, theseresults further confirm that IL-15 functions as a selectable marker genein cytotoxic lymphocytes genetically modified with an IL-15 expressionconstruct.

Materials and Methods (of Example 1)

Cells and culture conditions. Human NK cells were maintained in X-VIVO10medium supplemented with 5% human plasma and 100 IU/mL IL-2. IL-15expressing NK cells were cultured in X-VIVO10 medium supplemented with5% human plasma in the absence of exogenous cytokines. Murine CTL weremaintained in RPMI 1640 medium supplemented with 10% fetal bovine serum,2 mM L-glutamine, 100 U/mL penicillin, 100 μg/ml streptomycin, 50 μMβ-mercaptoethanol and 50 IU/mL IL-2. IL-15 expressing CTL were culturedin the same medium in the absence of exogenous cytokines.

Construction of IL-15 expression vector. The lentiviral transfer vectorSIEW was used as basis for the construction of different IL-15 encodingvectors for expression in lymphocytes. Two types of bicistroniclentiviral transfer vectors were constructed that employ IL-15 as aselectable marker. First, IL-15 cDNA was inserted into the SacIIrestriction site of SIEW resulting in a vector encoding IL-15 under thecontrol of the Spleen Focus Forming Virus promoter followed by aninternal ribosome entry site (IRES) sequence and enhanced greenfluorescent protein (EGFP) cDNA. For a second type of bicistronicvector, the IRES sequence was amplified by PCR using SIEW plasmid DNA astemplate and the oligonucleotide primers:

5′-SacII-IRES [SEQ ID NO. 5]5′-AAACCGCGGAAAAAAACTGGCAAGAACTGACGAGTTCGTATTCCC GGCCGCAGCC-3′ and3′-XbaI-IRES [SEQ ID NO. 6]5′-AAATCTA-GAAAACCACGTCCCCGTGGTTCGGGGGGCCTAG-3′.

The resulting PCR product was digested with SacII and XbaI and subclonedinto pBluescript SK(−) (pBSK) to generate the plasmid pBSK-IRES. IL-15cDNA was amplified using the oligonucleotide primers:

5′-XbaI-IRES-IL15 [SEQ ID NO. 7]5′-AAATCTAGAATGAGAATTTCGAAACCACATTTGAG-3′ and 3′-SwaI-IRES-IL15[SEQ ID NO. 8] 5′-AAAAAATTTAAATATTATCAAGAAGTGTTGATGAACATTTGG-3′.

The resulting PCR product was digested with XbaI and SwaI and ligatedinto XbaI and EcoRV digested pBSK-IRES to generate pBSK-IRES-IL-15. Thenthe IRES-IL-15 expression cassette was isolated by digestion with SacIIand HindIII, and ligated into SacII and Swal digested lentiviraltransfer vector SIEW. The resulting vector contains a SacII restrictionsite for insertion of a gene of interest, followed by an IRES sequenceand IL-15 cDNA as a selectable marker. Chimeric antigen receptorsequences were inserted via SacII resulting in vectors encoding a CAR asa gene of interest, followed by an IRES sequence and IL-15 cDNA forselection.

Production of VSV-G pseudotyped vectors in 293T cells. Vector particleswere generated by transient transfection of 4×10⁶ HEK-293T cells with athree plasmid system consisting of the packaging plasmid coding for theVSV-G envelope protein (pMD-VSVG), the glycoprotein expression plasmidencoding gag and pol (8.91), and the transfer plasmid carrying the geneof interest. Cells were transfected by calcium phosphate transfectionusing a total of 20 μg plasmid DNA consisting of 6.5 μg gag pol, 3.5 μgVSV-G, and 10 μg of transfer plasmids. DNA-calciumphosphate-precipitates were added dropwise to cell monolayers, and 10 mMchloroquine were added. Cell culture supernatants containing pseudotypedlentiviral vector particles were harvested 48 h later. Supernatants weresterile filtered (0.45 μm filter) and directly used for transduction ofNK cells and CTL.

Lentiviral transduction. For transduction, 5×10⁵ NK cells or CTL wereseeded into a single well of a 6 well plate. Vector particles were addedto the cells in the presence of 8 μg/mL polybrene and centrifuged for 60min at 1800 rpm at 32° C. 48 h after transduction the cells wereanalyzed by FACS for EGFP and CAR expression.

Flow cytometric analysis. For analysis of EGFP expression, transduced NKcells were harvested, washed once in FACS buffer (DPBS, 3% FCS),resuspended in 250 μL FACS buffer, and directly analyzed using aFACSCanto flow cytometer (BD Biosciences). Untransduced cells served ascontrol. For analysis of CAR expression, washed NK cells were incubatedwith 1 μg CAR-specific antibody (EpCAM-specific CAR) or 1 μg ErbB2-Fcfusion protein (R&D Systems) (ErbB2-specific CAR) for 1 h at 4° C. Thencells were washed and stained with a species-specific secondaryAPC-coupled antibody for 20 min at 4° C. Samples were washed in FACSbuffer and resuspended in 250 μl for FACS analysis using a FACSCantoflow cytometer (BD Biosciences). NK cells transduced with an IL-15expression construct served as control.

Cell growth kinetics. NK cells or CTL were seeded in triplicates in96-well plates at a density of 1×10⁴ cells/well in normal growth mediumwith or without addition of 100 IU/mL IL-2 (NK cells) or 50 IU/mL IL-2(CTL). The cells were incubated for up to 9 days at 37° C. in ahumidified atmosphere of 95% air, 5% CO₂. At different time points (days1, 3, 6, 9 for NK cells; days 2, 3, 4 for CTL) the relative number ofviable cells was determined in MTT metabolization assays. Ten μL of 10mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide] (Sigma, Deisenhofen, Germany) in DPBS were added to each well,and the cells were incubated for 4 hours. Cells were then lysed by theaddition of 90 μL 20% SDS in 50% dimethyl formamide, pH 4.7. Aftersolubilisation, color development due to formation of a brown formazanproduct was quantified by determining the absorbance at 590 nm in amicroplate reader. Samples without cells served as blank.

FACS-based cytotoxicity assays. To investigate cytotoxic activity ofparental and genetically modified NK cells (effector cells, E) towardsdifferent tumor cell lines (target cells, T), a FACS-based cytotoxicityassay was used. Target cells were labeled with calcein violet AM(Molecular Probes, Invitrogen). Cells were harvested, counted and washedin calcein wash buffer (RPMI1640). The cell number was adjusted to 4×10⁶cells/mL, and 1.5 μL calcein violet AM dissolved in 42 μL DMSO wereadded to the cells. Staining of cells was performed for 30 min on ice.Then cells were washed three times with calcein wash buffer, and thecell number was adjusted to 5×10⁵ cells/mL. To test cytotoxic activityof genetically modified NK cells, effector and labeled target cells wereco-cultured at various effector to target (E/T) ratios. First, effectorcells were pelleted, counted and the cell number was adjusted to 5×10⁶cells/mL. Appropriate dilutions were prepared. For co-cultureexperiments target cells were resuspended in X-VIVO medium containing 5%human plasma without addition of exogenous cytokines. 100 μL targetcells were co-cultured with 100 μL effector cells at various E/T ratiosfor 2 h at 37° C. Then samples were washed once in FACS buffer.Spontaneous target-cell lysis was determined in samples only containinglabeled target cells. 250 μL propidium iodide solution (1 μg/mL) wereadded to the samples shortly before measurement. Cells were analyzed ina FACSCanto flow cytometer (BD Biosciences). The percentage of deadtarget cells was determined using FACSDiVa software (BD Biosciences).

IL-15 activity in culture supernatant of IL-15 expressing cytotoxiclymphocytes. For analysis of soluble IL-15 activity in the supernatantof NK cells transduced with a lentiviral vector encoding anEpCAM-specific CAR followed by an internal ribosome entry site and IL-15as a selectable marker, conditioned medium from 1×10⁶ gene-modified NKcells grown for three days in 10 mL of growth medium without IL-2 wascollected and sterile filtered. Subsequently, IL-2/IL-15-dependentmurine CTLL-2 cells (ATCC number TIB-214) were seeded in triplicates in96-well plates at a density of 1×10⁴ cells/well in conditioned culturesupernantant of gene-modified NK cells. CTLL-2 cells grown in culturesupernatant of untransduced NK cells left in medium lacking IL-2, andCTLL-2 cells grown in regular growth medium with or without the additionof 50 IU/mL IL-2 served as controls. The cells were incubated for up to5 days at 37° C. in a humidified atmosphere of 95% air, 5% CO₂. Atdifferent time points (days 1, 2, 5) the relative number of viable cellswas determined in MTT metabolization assays as described above for thedetermination of cell growth kinetics.

REFERENCES

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The invention claimed is:
 1. An isolated transgenic mammalian cytotoxicT lymphocyte (CTL) or natural killer (NK) cell expressing IL-15 as aselectable marker for gene transfer and co-expressing at least oneprotein of interest, said cell comprising a lentiviral vector encodingIL-15 and the at least one protein of interest, wherein the protein ofinterest is a protein other than IL-15, and wherein the protein ofinterest is a protein of therapeutic interest, wherein the expression ofthe IL-15 as a selectable marker results in survival or growth of themammalian CTL or NK cell in the absence of exogenously added IL-15and/or IL-2, wherein the IL-15 has the amino acid sequence of SEQ ID NO.1 or an amino acid sequence that has at least 95% sequence identity toSEQ ID NO. 1, or the IL-15 is encoded by the nucleotide sequence of SEQID NO. 2 or a nucleotide sequence with at least 95% sequence identity toSEQ ID NO. 2, and wherein the expression of the IL-15 does not result inthe secretion of IL-15 into the culture supernatant in amountssufficient to support survival and growth of cells that are nottransformed or transduced with the IL-15 and/or are not expressing theIL-15 themselves.
 2. The transgenic mammalian cell according to claim 1,wherein the protein of therapeutic interest is a chimeric antigenreceptor (CAR), wherein the CAR comprises the following: (i) a signalpeptide; (ii) a target specific recognition domain that binds anantigen, receptor, peptide ligand or protein ligand of the target,wherein the target is a cell or a virus; (iii) a linker region,connecting domain (ii) and domain (iv); and (iv) an effector domaincomprising a transmembrane region and one or more intracellularsignaling domains.
 3. The transgenic mammalian cell according to claim1, wherein the IL-15is encoded by a nucleotide sequence encoding humanIL-15 having the amino acid sequence of SEQ ID NO. 1, a nucleotidesequence comprising the nucleotide sequence of human IL-15 transcriptvariant 3 of SEQ ID NO. 2, or a complementary sequence thereof, or acodon-optimized sequence thereof, or a nucleotide sequence encoding anamino acid sequence with at least 95% sequence identity to the aminoacid sequence of SEQ ID NO. 1, or a nucleotide sequence with at least95% sequence identity to the nucleotide sequence of SEQ ID NO.
 2. 4. Apharmaceutical composition comprising the cell of claim 1, and apharmaceutical carrier.
 5. The transgenic mammalian cell, according toclaim 2, wherein the CAR comprises an antigen binding domain derivedfrom an antibody against an antigen of the target, or a peptide thatbinds an antigen of the target, or a peptide or protein that binds anantibody that binds an antigen of the target, or a peptide or proteinligand that binds a receptor on the target, or a domain derived from areceptor that binds a peptide or protein ligand on the target, and/or,where domain (ii) of the CAR binds an antigen of the target, wherein theantigen is a tumor-associated surface antigen, a lineage-specific ortissue-specific surface antigen or a virus-specific surface antigen;and/or, where domain (ii) of the CAR comprises an antigen bindingdomain, which is derived from an antibody or a single chain variablefragment (scFv).
 6. The transgenic mammalian cell, according to claim 2,wherein the linker region (iii) of the CAR comprises a hinge regionderived from the human CD8 alpha-chain and/or wherein the effectordomain (iv) of the CAR comprises the zeta-chain of the human CD3 complexof the T-cell receptor or a CD28-CD3 zeta-chain fusion.